Student Code

: ____________

20th INTERNATIONAL BIOLOGY OLYMPIAD

12th 19th July 2009
Tsukuba, JAPAN

PRACTICAL TEST 2 / 2

BIOCHEMISTRY /
Total Points: 100 /
Duration: 90 minutes / :90

Dear Participants,
,

In this test, you have been given the following 2 tasks:

o Task 1: Measurement of acid phosphatase activity
o Task 2: Protein determination

(70 points)

(30 points)

:
o 1: (70 points)
o 2:

You must write down your results and answers in the ANSWER SHEET. Answers
written in the Question Paper will not be evaluated.
.

Please make sure that you have received all the materials and equipment listed for each
task. If any of these items are missing, please raise your hand.

.

At the end of the test, put the Answer Sheet and Question Paper in the envelope. The
supervisor will collect this envelope.

Good Luck!!

 !!

How to use the spectrophotometer

1. The screen of spectrophotometer (Shimadzu UVmini-1240) must show 400 nm (Fig. 1).
If not, raise your hand. ABS value shown may not be 0.000.
400 nm ( 1).
.
0.000.
2. Fill a plastic semi-micro cuvette with distilled water (DW) at least up to the shoulders
inside (Fig. 2)
( , ...)
(DW) .
(( 2).
3. Insert the cuvette into the cuvette holder of the instrument, with the transparent surfaces
facing to the left and right (Fig. 3).

. !!! /
(
) ( 3).
4. Shut the lid (Fig. 4).
( 4).
5. Press AUTO ZERO button (Fig. 5). By this manipulation, the instrument regards the
level of absorbance by the cuvette plus water as zero.

 AUTO ZERO
. ( 5).
6. Now, you are ready to measure absorbance of samples.

7. Replace the water with a sample solution and read an ABS value after the lid is shut. The
absorbance is caused by solutes in the sample solution.
, (
)
ABS, ,
.
.
8. You do not have to wash the cuvette after every measurement, if you measure a series of
samples from the dilute to the concentrated.
,

.
.

Fig. 1 1

Shoulders

Fig. 2 2
Lid

Light

Fig. 3 3

Cuvette holder

Fig. 4

Fig. 5 5

Introduction

Acid phosphatase liberates phosphate ions from phosphorylated molecules under acidic
conditions. The purpose of this experiment is to determine the specific activity of the acid
phosphatase. You will measure activities of the acid phosphatase using a crude extract from
potato in Task 1, and determine a protein concentration of the crude extract in Task 2.
Specific activity, which is the activity per unit time per unit weight of protein, is obtained
from Tasks 1 and 2. Specific activity is an index of purity; it increases as the enzyme is
purified.
PO4 3-
.
. 1


2.
, :
.
1 2.

. -

Caution

1.

You will be handling small amounts of toxic substances (p-nitrophenol and

NaOH).You can choose to wear disposable gloves and safety goggles in the
experiments if you like.
(p-nitrophenol /
NaOH)

2.

In calculations where answers to previous questions are needed, partials marks will be
given if calculated formulas are correct, even if answers are incorrect.

,

Materials and Equipments

Quantity

1. Spectrophotometer -

2. Micropipettes (P1000)- 1000 l

3. Micropipettes (P200) - 200 l

4. Tips (one box each for P1000 and P200)

( P1000 P200
5. Plastic cuvette


6. Test tube holder that accommodates 6-1 to 6-7

:
6-1 Crude extract of acid phosphatase 1x enzyme

(4 ml in a 15-ml plastic ube) (4 ml 15-ml . )
6-2 0.5 M Na acetate buffer (pH 5.6)

0.5 M / Na acetate (pH 5.6)

(2 ml in a 15-ml plastic tube) (2 ml 15-ml . )
6-3 5 mM pNPP ( ) (4 ml in a 15-ml plastic tube)

(4 ml 15-ml . )
6-4 0.5 M NaOH (8 ml in a 15-ml plastic tube) 8ml .

15-ml
6-5 3% NaCl (10 ml in a 15-ml plastic tube) 10ml .

15-ml
6-6 glass Test tubes/

Task 1 1 (70 points): Measurement of acid phosphatase activity /

The activity of acid phosphatase is measured by an enzymatic reaction that converts pnitrophenyl phosphate (pNPP) to p-nitrophenol (pNP), liberating phosphate. The product,
pNP, absorbs light whose wavelength is 400 nm with an absorption coefficient(400 nm) of

19000 M-1 cm-1 at extremely alkaline pH. Reaction mixture for an acid phosphatase is
slightly acidic.
Thus, it must be alkalinized for quantification of pNP. In Task 1, you will measure a time
course of the reaction and obtain absorbance change per minute that is caused by 1 ml of
crude extract. The absorbance change is converted to concentration change by using 400 nm.
Then, you will calculate a mol number of pNP molecules produced during the reaction by
multiplying the concentration change by a volume of sample that is subjected to the
measurement of absorbance.

(pNPP) () (pNP) ()
. , p- (pNP)
400 nm, 400 nm
19000 M-1 cm-1 pH.
,
, pNP, ,
.

1 ml .
.
:
A, absorbance. ( ,
)
, absorption coefficient (M-1 cm-1).

C, concentration (M=mol litre-1 )

L, light path length (cm) (

)
I0, intensity of incident light . . ()
I, intensity of transmission light . (), .
( )

Absorbance (A) is a physico-chemical property of solution that expresses to what extent a solute
absorbs light at a specific wavelength.
Absorbance is in proportion to concentration (C) and light path length (L). The constant in the
equation is a value characteristic to the solute, and is termed the absorption coefficient (). Thus, the
relationship is formulated as A= C (M=mol litre-1) L (cm). Absorbance can be converted to
concentration, since is given and L is 1 cm in this experiment. The dimension of is M-1 cm-1,
because absorbance is an absolute number without units.

.
(C) (L) .

. : A= C L. (ABS)
!!! (mol / Litre)
() (mol/L ) (mol.litre-1 )
pNP =19000 mol. Litre-1, L 1 cm.
Two enzyme concentrations are to be examined in Task 1. Find the test tube on which 1x enzyme
is labeled, which contains a crude extract of acid phosphatase. Next, find the 15-ml tube that
contains 3% NaCl and remove 1 ml from that using the p1000 micropipette. Now you have a
tube containing 9 ml of 3% NaCl. To this tube add 1 ml of the 1x enzyme solution to it by
using a micropipette, which makes 0.1x enzyme solution. Re- Label the tube as 0.1x

enzyme. Label each tube with an enzyme concentration and a reaction time as follows.

Next, find 6 empty glass test tubes.

-
1. 1x enzyme
.
15-ml 3% NaCl. 1000
1 ml 10ml NaCl. 9 ml NaCl.
9 ml 1ml 1x enzyme.
10
0.1x enzyme ( x
). -
0.1x enzyme 3% NaCl.
6 .

:

0.1x, 20 min
1x, 20 min
0.1x, 10 min
1x, 10 min
0.1x, 1 min
1x, 1 min

Q.1.1. (10 points) First, make an experimental schedule in order to perform all reactions, by
describing start () and stop () signs for each reaction in the table in the Answer Sheet,
allowing at least 1 min between the beginning of each reaction. An example for the reaction
of 0.1x, 20 min has been described in the table in the Answer Sheet.

() , ()
, . 1
. 0.1x, 20
min .

Q.1.2. (15+10 points) Perform the enzymatic reactions according to the protocol described
below and the schedule you made in Q.1.1. Use a new pipette tip in every manipulation.
Agitate a mixture by tapping a test tube immediately after an addition. After you perform all
the reactions, measure A400 of the samples. Write the obtained values in the table in the
Answer Sheet, and plot them in the graph. Please note that since water has been used as a
blank the line will not pass through zero (0) on the Y axis.

,
Q.1.1.
.
.
.
A400 ( ).


. !
( )
(0).

Protocol for measurement of acid phosphatase activity/

.
1. Mix 0.12 ml of 0.5 M Na acetate buffer (pH 5.6) and 0.24 ml of 5 mM pNPP in a test
tube. Start the reaction by adding 0.24 ml of an enzyme solution.
0.12 ml 0.5 M (pH 5.6)
0.24 ml pNPP 5 mM .
0.24 ml ( ,

.
2. After the reaction times of 1, 10, and 20 min, respectively, stop the reaction by adding
0.6 ml of 0.5 M NaOH. NaOH stops the reaction and converts the pNP produced into
a yellow-colored (A400-absorbing) form.
1, 10 20
0.6 ml NaOH 0.5 M. To NaOH
pNP ( ),
400 nm A400nm,
3. After all reactions are stopped, measure A400 of the samples.
, S 400 nm
(A400) . .
( 0.1 )

( 1 ).
. ,
. .
Assay of potato acid phosphatase

0.5 M Na acetate buffer(pH 5.6) /0.5 M (pH 5.6)

0.12

ml

5 mM pNPP

0.24

ml

Enzyme/

0.24

ml

0.5 M NaOH

0.6

ml

Sum/

1.2

ml

Q.1.3. (15 points) Which enzyme concentration gave better linearity in the relationship
between time and A400? Circle the correct one on the Answer Sheet. Read the slope of this
straight line from the graph.
t
A400? .
. (
.

Q.1.4. (5 points) Using the slope obtained in Q. 1.3, calculate the activity in the form of
A400 change per min per 1 ml of an enzyme solution of concentration 1x. Your answer should
be written with your calculations and the appropriate unit in the Answer Sheet. The length of
the light path (L) is 1cm
Q. 1.3,
- () A400

 ml (min-1 ml-1) 1x enzyme.

.

Q.1.5. (5 points) Convert the absorbance change obtained in Q.1.4 to a concentration change
by using the 400 of pNP (19000 M-1 cm-1). Your answer should be written as concentration
change per min per 1 ml of 1x enzyme solution in the Answer Sheet.
Q.1.4
. {400 pNP (19000 M-1 cm1

)} .

: pNP, ml ( min-1.ml-1)
1x enzyme.

Q.1.6. (5 points) Convert the concentration change obtained in Q.1.5. to a change in number
of moles of pNP. Your answer should be written with your calculations in moles min per 1 ml
of 1x enzyme solution in the Answer Sheet.
Q.1.5
pNP. {400 pNP
(19000 M-1 cm-1)} .
: moles, ml ( moles min-1.ml-1)
1x enzyme.

Q.1.7. (5 points) Calculate the total activity (in mol.min-1 )in 4 ml of 1x enzyme that was
initially given.
(mol.min-1 ) 4 ml
1x enzyme .

Task 2 (30 points) 2 (30 )

Protein determination/
Protein concentration is determined by using a standard protein such as bovine serum
albumin (BSA). In Task 2, you will determine a BSA-equivalent concentration of the 1x
enzyme solution by the Bradford method. The Bradford method takes advantage of an
increase in absorption of Coommassie Brilliant Blue at 595 nm when it is bound to protein.

(BSA). ,
1x enzyme BSA,
Bradford.
=595 nm , Coommassie Brilliant Blue

595 nm

By diluting a concentrated BSA solution (0.4 mg protein. ml-1) with 3% NaCl, a 1/2dilution series was made (0.4, 0.2, 0.1, and 0.05 mg protein . ml-1). The dilution series of
BSA and the 0.1x enzyme solution, which was made in Task 1, were all similarly treated
with dye. Optical density at 595 nm (OD595) of these samples was measured and recorded in
the table below.
BSA 5 (0.4, 0.2, 0.1, and
0.05 mg. ml-1)
!

 0.1x enzyme.
, :
Table
S a m p le /

' 0 . 1 x e n z y m e ' s o lu ti o n
/ 0.1x ( 10

,
)

[B S A ]

O D 595

( m g m l - 1 )
0

0.0 00

0 .05

0.0 70

0 .1

0.1 43

0 .2

0.2 61

0 .4

0.5 21

0.1 80

Optical density (OD), a measure of the extent to which a substance transmits light or the
absorbance of suspension of particles.

Q.2.1.(10 points) Plot OD595 against BSA concentration in the graph in the Answer Sheet
and depict an approximate straight line.
.
OD595 BSA.
, ,
. .

Q.2.2.(10 points) Estimate a protein concentration of the 0.1x enzyme solution from the
graph, and obtain the protein concentration of the 1x enzyme solution.
Q.2.1.
0.1x enzyme 1x
enzyme

Q.2.3.(10 points) Calculate the specific activity (activity per min per mg protein) of the 1x
enzyme solution. Your answer should be written with your calculations and the unit per min
per mg protein in the Answer Sheet.
(
mg , 1x enzyme.
mg
.